1. Field of the Invention
The present invention relates generally to atomic force microscopy (AFM) and to methods for using atomic force microscopy to measure intramolecular forces such as those associated with the secondary structure of RNA, DNA and proteins.
2. Description of the Related Art
In conventional atomic force microscopy, a sample is scanned with a fine-tipped probe mounted on a cantilever, and deflections of the probe tip as it passes across the sample are measured to determine the topography of the sample. Deflections in the probe tip as it passes along the sample surface may be monitored by various methods, including optical reflection, interferometry and piezoelectric strain gauge methods. See, for example, U.S. Pat. No. Re. 33,387 to Binnig, U.S. Pat. No. 5,144,833 to Amer et al, U.S. Pat. No. 5,463,897 to Prater et al, U.S. Pat. No. Re. 34,489 to Hansma et al and U.S. Pat. No. 5,260,824 to Okada et al, all of the above incorporated herein by reference.
In recent years, atomic force microscopy has been used to measure interfacial properties and intermolecular interactions such as elasticity, friction, adhesion, receptor/ligand interactions and antibody/antigen interactions between individual molecules. The use of atomic force microscopy to study intermolecular forces is described, for example, in the following patents, patent applications and publications, incorporated herein by reference: U.S. Pat. No. 5,363,697 to Nakagawa; U.S. Pat. No. 5,372,930 to Colton et al; Florin E. -L. et al, "Adhesion Forces Between Individual Ligand-Receptor Pairs" Science 264 (1994). pp 415-417; Lee, G. U et al, "Sensing Discrete Streptavidin-Biotin Interactions with Atomic Force Microscopy" Langmuir, vol. 10(2), (1994) pp 354-357; Dammer U. et al "Specific Antigen/Antibody Interactions Measured by Force Microscopy" Biophysical Journal Vol. 70 (May 1996) pp 2437-2441; Chilikoti A. et al, "The Relationship Between Ligand-Binding Thermodynamics and Protein-Ligand Interaction Forces Measured by Atomic Force Microscopy" Biophysical Journal Vol. 69 (November 1995) pp 2125-2130; Allen S. et al, "Detection of Antigen-Antibody Binding Events with the Atomic Force Microscope" Biochemistry, Vol. 36, No. 24 (1997) pp7457-7463; and Moy V. T. et al, "Adhesive Forces Between Ligand and Receptor Measured by AFM" Colloids and Surfaces A: Physicochemical and Engineering Aspects 93 (1994) pp 343-348, and U.S. patent application Ser. No. 09/074,541, filed May 8, 1998 for "APPARATUS AND METHOD FOR MEASURING INTERMOLECULAR INTERACTIONS BY ATOMIC FORCE MICROSCOPY" by John-Bruce DeVault Green and Gil U Lee. If interactions between molecules are studied in liquids, the experimental conditions, such as pH, buffer/ionic concentration, buffer/ionic species, etc. may be varied to determine the effect that these have on the forces of interaction.
Atomic force microscopy has great potential for use in measuring intramolecular forces such as those associated with the secondary or tertiary structure of RNA, DNA and proteins. Modern AFM instruments have sufficient sensitivity so that when a molecule such as DNA, RNA or a protein is pulled on, the forces holding together the secondary or tertiary structure of the molecule can be measured. See, for example, G. Lee et al, "Direct Measurement of the Forces Between Complementary Strands of DNA", Science, 1994, 266, pp 771-773, incorporated herein by reference. This article describes an experiment wherein poly-cytosine molecules were attached to the surfaces of a cantilever and a substrate stage of an atomic force microscope. Poly-inosine having an average base length of 160 bases was allowed to hybridize with the poly-cytosine so that the poly-inosine bridged the gap between the cantilever and the substrate. The force versus distance curve plot that was recorded as the surfaces were brought apart and separated showed a long-range cohesive force that can be attributed to the effects of intramolecular forces within the poly-inosine chain. See also M. Reif et al, "Reversible Unfolding of Individual Titin Immunoglobulin Domains by AMF," Science, 1997, 276, pp 1109-1112, incorporated herein by reference. The use of atomic force microscopy to sequence DNA has been described, for example, in Besimon, et al, PCT Application WO94/23065, published on Oct. 13, 1994.
However, the equipment and techniques currently used for atomic force microscopy are not well suited for repetitive measurements of intramolecular forces such as may be required to generate statistically valid data. In particular, chemically modified cantilever probe tips are fragile and easily damaged or inactivated. In a typical chemically modified cantilever probe tip, only the molecules that are bound to the very apex of the tip are available for interacting with a substrate. The crucial area of the probe tip is typically very small and the number of molecules bound thereon is very few; if anything happens to damage those few molecules or to block access to, or to otherwise inactivate that small area of the probe tip, then the probe tip is rendered useless and must be replaced. Replacing the probe tip usually requires replacing the entire cantilever, a procedure that is expensive and time-consuming. Moreover, the typical cantilever probe tip has room for only one type of grasping molecule to be immobilized on the tip, so the cantilever must be replaced or modified whenever it is desired to use a different grasping molecule.